Chiral N-acyl-5,6,7(8-substituted)-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazines as selective NK-3 receptor antagonists, pharmaceutical composition, methods for use in NK-3 receptor mediated disorders and chiral synthesis thereof

Abstract

The present invention relates to novel compounds of Formula I and their use in therapeutic treatments. The invention further relates to a novel chiral synthesis of 5,6,7,(8-substituted)-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazines using N-sp3 protective groups. The invention also provides intermediates for use in the synthesis of compounds of Formula I. ##STR00001##

Claims

1. A compound of Formula D ##STR00229## or a salt or solvate thereof, wherein the solid line with a star indicates that the individual enantiomers are meant, excluding racemic mixtures thereof; R.sup.1 is linear or branched C1-C4 alkyl or C3-C4 cycloalkyl, each of said alkyl or cycloakyl groups being optionally substituted by one or more group(s) selected from halo or esters; PG is a N-sp.sup.3 protective group; and R.sup.10 is C1-C2 alkyl.

2. The compound of claim 1, having Formula D-1 ##STR00230## or a salt or solvate thereof, wherein the solid line with a star indicates that the individual enantiomers are meant, excluding racemic mixtures thereof; R.sup.12, R.sup.12, R.sup.13, R.sup.13 and R.sup.14are H, and R.sup.12 and R.sup.12, R.sup.13 and R.sup.13 are H, or R.sup.12 and R.sup.14 are methoxy and R.sup.12, R.sup.13 and R.sup.13 are H, or R.sup.12, R.sup.12 and R.sup.14 are methoxy and R.sup.13 and R.sup.13 are H.

3. The compound of claim 1, wherein R.sup.1 is C1-C2 alkyl optionally substituted by one ester group.

4. The compound of claim 1 being (R)-1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine or (S)-1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows X-ray crystal structure of compound n.sup.o 1 (thermal displacement ellipsoids drawn at the 50% probability level).

(2) FIG. 2 shows X-ray crystal structure of compound n.sup.o 19 (thermal displacement ellipsoids drawn at the 50% probability level).

(3) FIG. 3 shows the effects of a single intravenous 10 mg/kg dose of compound no 1 on lutenizing hormone (LH) plasma levels in castrated male, Sprague-Dawley rats measured 1 hour after dosing. LH levels are expressed as meansS.E.M. The vehicle is 9% 2-hydroxypropyl--cyclodextrin/H.sub.2O (w/w). Vehicle, N=10 rats; Compound n.sup.o 1, N=9 rats.

(4) FIG. 4 shows the effects of a single intravenous 10 mg/kg dose of compound n.sup.o 19 on lutenizing hormone (LH) plasma levels in castrated male, Sprague-Dawley rats measured 1 hour after dosing. LH levels are expressed as meansS.E.M. The vehicle is 9% 2-hydroxypropyl--cyclodextrin/H2O (w/w). Vehicle, n=5 rats; Compound n.sup.o 19, n=5 rats.

(5) FIG. 5 shows the effects of a single intravenous 50 mg/kg dose of compound n.sup.o 1 on testosterone plasma levels in male, Sprague-Dawley rats. N=3 rats per treatment group. Testosterone levels were measured just prior to dosing and at times 1, 5, 15, 90, 150, 210 min after dosing to derive a time-response curve. Data are expressed as the testosterone area under the curve (AUC)S.E.M. The vehicle is 9% 2-hydroxypropyl--cyclodextrin/H.sub.2O (w/w).

EXAMPLES

Chemistry Examples

(6) All reported temperatures are expressed in degrees Celsius ( C.); all reactions were carried out at room temperature (RT) unless otherwise stated.

(7) All reactions were followed by thin layer chromatography (TLC) analysis (TLC plates, silica gel 60 F.sub.254, Merck) was used to monitor reactions, establish silica-gel flash chromatography conditions. All other TLC developing agents/visualization techniques, experimental set-up or purification procedures that were used in this invention, when not described in specific details, are assumed to be known to those conversant in the art and are described in such standard reference manuals as: i) Gordon, A. J.; Ford, R. A. The Chemist's CompanionA Handbook of Practical Data, Techniques, and References, Wiley: New York, 1972; ii) Vogel's Textbook of Practical Organic Chemistry, Pearson Prentice Hall: London, 1989.

(8) HPLC-MS spectra were typically obtained on an Agilent LCMS using electropsray ionization (ESI). The Agilent instrument includes an autosampler 1200, a binary pump 1100, an ultraviolet multi-wavelength detector 1100 and a 6100 single-quad mass-spectrometer. The chromatography column used was Sunfire 3.5 m, C18, 3.050 mm in dimensions.

(9) Eluent typically used was a mixture of solution A (0.1% TFA in H.sub.2O) and solution B (0.1% TFA in MeCN).

(10) Gradient was applied at a flow rate of 1.3 mL per minute as follows: gradient A: held the initial conditions of 5% solution B for 0.2 min, increased linearly to 95% solution B in 6 min, held at 95% during 1.75 min, returned to initial conditions in 0.25 min and maintained for 2.0 min; gradient B: held the initial conditions of 5% solution B for 0.2 min, increased linearly to 95% in 2.0 min, held at 95% during 1.75 min, returned to initial conditions in 0.25 min and maintained for 2 min.

(11) Determination of chiral purity was made using chiral HPLC that was performed on an Agilent 1100 (binary pump and a ultraviolet multi wavelength detector) with manual or automatic (Autosampler 1100) injection capabilities. Columns used were CHIRALPAK IA 5 m, 4.6250 mm or CHIRALPAK IB 5 m, 4.6250 mm in isocratic mode. Choice of eluent was predicated on the specifics of each separation. Further details concerning the chiral HPLC methods used are provided below.

(12) Method A: column CHIRALPAK IA 5 m, 4.6250 mm, eluent: EtOAc plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 nm; column at RT, eluent was used as sample solvent.

(13) Method A: column CHIRALPAK IA 5 m, 4.6250 mm, eluent: EtOAc plus 0.1% of DEA, flow rate: 1.5 mL per minute; UV detection at 254 nm; column at RT, eluent was used as sample solvent.

(14) Method B: column CHIRALPAK IA 5 m 4.6250 mm, eluent: hexane/isopropanol/dichlormethane (3:1:1 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 nm, column at RT, eluent was used as sample solvent.

(15) Method B: column CHIRALPAK IA 5 m 4.6250 mm, eluent: hexane/isopropanol/dichlormethane (3:1:1 v/v) plus 0.1% of DEA, flow rate: 1.5 mL per minute; UV detection at 254 nm, column at RT, eluent was used as sample solvent. Method C: column CHIRALPAK IB 5 m 4.6250 mm, eluent: hexane/ethanol (7:3 v/v) plus 0.1% of DEA, flow rate: 1.0 mL min.sup.1, mL per minute; UV detection at 254 nm, column at RT, eluent was used as sample solvent.

(16) Method C: column CHIRALPAK IA 5 m 4.6250 mm, eluent: hexane/ethanol (1:1 v/v) plus 0.1% of DEA, flow rate: 1.0 mL per minute; UV detection at 254 nm, column at RT, eluent was used as sample solvent.

(17) Preparative HPLC purifications were typically carried out on a Waters FractionLynx instrument. This instrument consists of a fraction collector, a 2767 sample manager, a pump control a module II, a 515 HPLC pump, a 2525 binary gradient module, a switching valve, a 2996 photodiode array detector and a Micromass ZQ mass spectrometer. The chromatography column used was Waters Sunfire 5 m, C18, 19100 mm, or XBridge 5 m, C18, 19100 mm depending on the type of eluent system employed, i.e. low pH or high pH conditions.

(18) For high-pH HPLC purifications, eluent typically consisted of a mixture of solution A (0.04 M ammonium bicarbonate in H.sub.2O plus 0.1% of conc. NH.sub.4OH) and solution B was MeCN. The gradient was adapted depending on the impurity profile in each sample purified, thereby allowing sufficient separation between the impurities and the desired compound.

(19) Chiral preparative HPLC purifications were performed on an Agilent 1200 instrument (preparative pump 1200 and ultraviolet multi wavelength detector 1200) with manual injection. The chiral columns used are as follows: CHIRALPAK IA 5 m, 20250 mm, CHIRALPAK IA 5 m, 10250 mm or a CHIRALPAK IB 5 m, 10250 mm. All chiral HPLC methods were employed in an isocratic mode. The eluent mixture was selected based on the analytical chiral HPLC experiment (see above) that provided the best chiral separation.

(20) .sup.1H (300 MHz) and .sup.13C NMR (75 MHz) spectra were recorded on a Bruker Avance DRX 300 instrument. Chemical shifts are expressed in parts per million, (ppm, units). Coupling constants are expressed in Hertz (Hz). Abbreviations for multiplicities observed in NMR spectra are as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), br (broad).

(21) Solvents, reagents and starting materials were purchased and used as received from commercial vendors unless otherwise specified.

(22) The following abbreviations are used:

(23) Boc: tert-butoxycarbonyl,

(24) Cpd: compound,

(25) DCM: Dichloromethane,

(26) DEA: diethylamine,

(27) DMA: N,N-dimethylaceetamide,

(28) DMB: 2,4-dimethoxybenzyl,

(29) DMB-CHO: 2,4-dimethoxybenzaldehyde,

(30) DMF: N,N-dimethylformamide,

(31) ee: Enantiomeric excess,

(32) eq: Equivalent(s),

(33) Et: Ethyl,

(34) EtOAc: Ethyl acetate,

(35) EtOH: Ethanol,

(36) g: Gram(s),

(37) h: Hour(s),

(38) IPA: isopropanol,

(39) L: Liter(s),

(40) MeOH: Methanol,

(41) L: Microliter(s),

(42) mg: Milligram(s),

(43) mL: Milliliter(s),

(44) mmol: Millimole(s),

(45) min: Minute(s),

(46) NMM: N-methylmorpholine

(47) P: UV purity at 254 nm or 215 nm determined by HPLC-MS,

(48) PMB: 4-methoxybenzyl,

(49) PMB-CHO: 4-methoxybenzaldehyde,

(50) RT: Room temperature,

(51) tBu: tert-Butyl,

(52) TFA: trifluoroacetic acid,

(53) THF: Tetrahydrofuran,

(54) TLC: Thin layer chromatography,

(55) TMS: trimethylsylil,

(56) Y: Yield.

(57) The intermediates and compounds described below were named using ChemDraw Ultra version 12.0 (CambridgeSoft, Cambridge, Mass., USA).

I. Racemic Synthesis

I.1. General Synthetic Scheme for Racemic Synthesis

(58) Most compounds of the invention were synthesized using the methodology described in Scheme 1, which represents the racemic product synthesis. The racemic products were subjected to chiral HPLC for chiral separation.

(59) ##STR00195##

(60) The general synthetic scheme comprises the following steps:

(61) Step 1: Ketopiperazine 1.1 was protected and converted to iminoether 1 by using the Meerwein reagent (Et.sub.3OBF.sub.4).

(62) Step 2: Ester 2.2 was subsequently converted to acyl hydrazide 2. Ester 2.2 may be obtained be esterification of acid 2.1.

(63) Step 3: Cyclodehydration between the acyl hydrazide 2 and the iminoether 1 furnished the protected triazolopiperazine 3.1. Thereafter, 3.1 was subjected to acidolytic deprotection to obtain 3.

(64) Step 4: The thus obtained triazolopiperazine intermediate 3 was acylated through reaction with the appropriate acid chloride 4.1 to obtain the racemic final target structure represented by the general Formula 4. The chiral final compounds were subsequently obtained by purification using preparative chiral HPLC.

I.2. Step 1: Protection and Conversion to Iminoether 1

Method A: Boc Protection and Conversion to Iminoether 1

(65) Method A is the procedure used for the synthesis of the iminoether intermediates 1 with a Boc protection and is detailed below:

(66) ##STR00196##

(67) Method A is illustrated by the synthesis of intermediates 1a and 1b wherein R1 is H and Me respectively.

Synthesis of tert-butyl 3-ethoxy-5,6-dihydropyrazine-1(2H)-carboxylate 1a

(68) ##STR00197##

(69) To a pre-made solution of triethyloxonium tetrafluoroborate (2.3 g, 0.012 mol) in anhydrous DCM (20 mL) was added 1.2a (2 g, 0.01 mol) at 0 C. After the addition was completed, the ice-bath was removed, and the reaction mixture was allowed to warm to RT and stirred for an additional hour (reaction progress monitored by LCMS). Upon completion of the reaction, a saturated solution of NaHCO.sub.3 (500 mL) was slowly added to the reaction mixture and it was stirred for 5 min. The organic layer was separated and the aqueous layer was further extracted with DCM (200 mL). The combined organic layers were subsequently washed with brine, dried over MgSO.sub.4, filtered and further dried in vacuo to obtain the title intermediate 1a as viscous yellow oil. Yield: 2.03 g (88%). .sup.1H NMR (CDCl.sub.3): : 4.1 (q, J=7.1, 2H), 3.85 (s, 2H), 3.5 (m, 1H), 3.35 (t, J=5.1, 2H), 1.45 (s, 9H), 1.3 (t, J=7.1, 3H).

Synthesis of tert-butyl 3-ethoxy-2-methyl-5,6-dihydropyrazine-1(2H)-carboxylate 1b

(70) ##STR00198##

Step 1: Synthesis of tert-butyl 2-methyl-3-oxopiperazine-1-carboxylate 1.2b

(71) NEt.sub.3 (20 mL, 145 mmol) was added to a solution of 3-methylpiperazin-2-one 1.1b (15 g, 131 mmol) in anhydrous DCM (200 mL) under N.sub.2 at RT. After 10 min stirring, the reaction mixture was cooled to 0 C. and Boc.sub.2O (33 g, 151 mmol). The reaction mixture was stirred at RT for 1 h and thereupon washed with 0.5M HCl (150 mL), brine (150 mL), dried over MgSO.sub.4, filtered and concentrated to constant weight furnishing 2.2 as yellow oil (20.2 g, 72%). LCMS: P=100%, retention time=2.0 min, (M+H-tBu).sup.+: 159

Step 2: Synthesis of tert-butyl 3-ethoxy-2-methyl-5,6-dihydropyrazine-1(2H)-carboxylate 1b

(72) To a solution of 1.2b (24 g, 87 mol) in anhydrous DCM (250 mL) at 0 C. under N.sub.2 atmosphere was added a pre-made solution of triethyloxonium tetrafluoroborate (19.92 g, 105 mmol) in anhydrous DCM (50 mL). The reaction mixture was allowed to warm to RT and stirred for 30 min whereupon saturated solution of NaHCO.sub.3 (400 mL) was added. The extracted aqueous layer was then washed with DCM (200 ml) and the combined organic extracts were subsequently washed with brine (300 mL), dried over MgSO.sub.4, filtered and further dried in vacuo to obtain the title intermediate 1b as colorless oil. (20.7 g, 98%). LCMS: P=98%, retention time=1.8 min, (M+H+H.sub.2O).sup.+: 261; .sup.1H-NMR (CDCl.sub.3): 4.30 (br, 1H), 4.11-4.01 (m, 2H), 3.84 (br, 1H), 3.48-3.40 (m, 2H), 2.90 (br, 1H), 1.32 (d, J=6.9, 3H), 1.26 (t, J=7.1, 3H).

Method B: Protection Using Benzyl Derivative Protecting Groups Such as DMB and Conversion to Iminoether 1

(73) Method B is the procedure used for the synthesis of the iminoether intermediates 1 with a benzyl derivative protecting group such as DMB and is detailed below:

(74) ##STR00199##

(75) Method B is illustrated by the synthesis of intermediates 1c and 1d wherein R1 is Me and the protecting group is DMB and PMB respectively.

Synthesis of 1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine 1c

(76) ##STR00200##

Step 1: Synthesis 4-(2,4-dimethoxybenzyl)-3-methylpiperazin-2-one 1.2c

(77) In a round-bottom flask, were sequentially introduced 3-methylpiperazin-2-one (10 g, 88 mmol), 2,4-dimethoxybenzaldehyde (16 g, 96 mmol), acetic acid (6.5 ml, 114 mmol) and sodium triacetoxyborohydride (22.3 g, 105 mmol) in commercial anhydrous acetonitrile (750 mL), at RT, under N.sub.2 atmosphere. The reaction was stirred at RT overnight. The reaction mixture was quenched carefully at 0 C. with saturated NaHCO.sub.3 solution (100 mL) until n.sup.o more bubbling was observed. Aqueous and organic layers were separated. The aqueous layer was extracted with EtOAc (3300 mL) and the combined organic layers were washed with brine, dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to afford the title compound as yellow oil. The crude compound was then purified on silica gel (DCM/MeOH: 98/2 to 95/5) to afford the desired product 1.2c as a pale yellow oil (20.6 g, 78 mmol, 89%). LCMS: P=97%, retention time=1.6 min, (M+H).sup.+: 265.

(78) In the case of PMB and TMB protection, 4-methoxybenzaldehyde or 2,4,6-trimethoxybenzaldehyde was used instead of 2,4-dimethoxybenzaldehyde to furnish 4-(4-methoxybenzyl)-3-methylpiperazin-2-one or 4-(2,4,6-trimethoxy benzyl)-3-methylpiperazin-2-one.

Step 2: 1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine 1c

(79) Oven-dried (115 C.) sodium carbonate (18.6 g, 98 mmol, 2.25 eq.) was placed in a 500 mL round-bottom flask. The round-bottom flask was backfilled with Ar and then capped with a rubber septum. A solution of 4-(2,4-dimethoxybenzyl)-3-methylpiperazin-2-one 1.2c (20.6 g, 78 mmol, 1 eq.) in anhydrous DCM (250 mL) was added, followed by triethyloxonium tetrafluoroborate (18.6 g, 98 mmol, 1.25 eq.) in one portion. Thereafter, the reaction mixture was stirred further at RT for 1 h whereupon the reaction mixture was diluted with water (250 mL). The aqueous layer was extracted with DCM (3150 mL). The organic layers were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude compound was then purified on silica gel (EtOAc) to afford the desired product 1c as orange oil. Yield: 13.2 g, 58%. LCMS: P=93%, retention time=1.8 min, (M+H+H.sub.2O).sup.+: 311; .sup.1H-NMR (CDCl.sub.3): 7.23 (d, J=8.8 Hz, 1H), 6.48 (d, J=8.8 Hz, 1H), 6.44 (s, 1H), 4.02 (m, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.86 (d, J.sub.AB=14.0 Hz, 1H), 3.46 (d, J.sub.AB=14.0 Hz, 1H), 3.44 (m, 2H), 3.10 (m, 1H), 2.79 (m, 1H), 2.32 (m, 1H), 1.35 (d, J=6.8 Hz, 3H), 1.24 (t, J=6.0 Hz, 3H).

(80) Starting step 2 from 4-(4-methoxybenzyl)-3-methylpiperazin-2-one allowed to isolate 1-(4-methoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine 1d. LCMS: P=95%, retention time=1.8 min, (M+H+H.sub.2O).sup.+: 281.

I.3. Step 2: Formation of Acyl Hydrazide 2

Method C: Acyl Hydrazide 2

(81) Method C is the procedure used for the synthesis of the acyl hydrazides 2 and is detailed below:

(82) ##STR00201##

(83) Method C is illustrated by the synthesis of intermediate 2a, 2k and 2r.

Synthesis of 2-methylthiazole-4-carbohydrazide 2a

(84) ##STR00202##

(85) In a 100 mL round-bottom flask equipped with a condenser, ethyl 2-methylthiazole-4-carboxylate 2.2a (10 g, 58.4 mmol, 1 eq.) was dissolved in anhydrous EtOH (25 mL) and treated at RT with hydrazine monohydrate (17.0 mL, 354.4 mmol, 6 eq.). The resulting yellow solution was heated at reflux temperature for 14 h. After allowing the reaction mixture to come to RT, the solution was concentrated under reduced pressure to afford 13.4 g of a brown oil. Co-evaporations using 3200 mL of a mixture of commercial anhydrous DCM:MeOH (1:1) were performed to remove residual water. The residue was then recrystallized from hot EtOH (60 mL). The obtained crystals were filtered and washed with cooled (0 C.) EtOH (230 mL). The orange solid was dried under vacuum for 1 h to afford 2a. Yield: 5.85 g, 64%. LCMS: P=100%, retention time=0.5 min, (M+H).sup.+: 158; .sup.1H-NMR (CDCl.sub.3): 8.32 (br, 1H), 7.96 (s, 1H), 4.07 (br, 2H), 2.70 (s, 3H).

(86) In one embodiment 1.2 to 20 equivalents of hydrazine hydrate was used to carry out this reaction using a temperature range from RT to reflux.

(87) In one embodiment, hydrazide 2 was recrystallized and/or precipitated.

(88) The following intermediates were also prepared from the ad hoc carboxylic acids, methyl or ethyl esters using General Method C:

(89) intermediate 2b: 2-trifluoromethylthiazole-4-carbohydrazide, methyl ester precursor was previously synthesized using conventional esterification method (such as TMS-C1 in methanol) from commercially available acid;

(90) intermediate 2c: 2-ethylthiazole-4-carbohydrazide;

(91) intermediate 2d: 2-vinylthiazole-4-carbohydrazide, tert-butyl 2-(4-(hydrazinecarbonyl)thiazol-2-yl)ethylcarbamate was used as precursor of the vinyl moiety, commercially available ethyl 2-(2-aminoethyl)thiazole-4-carboxylate dihydrochloride was previously Boc-protected and then esterified using conventional methods;

(92) intermediate 2e: 2-methyloxazole-4-carbohydrazide;

(93) intermediate 2f: 2-isopropyloxazole-4-carbohydrazide, ethyl ester precursor was previously synthesized from condensation of isobutyramide and ethyl 3-bromo-2-oxopropanoate according to WO2009/70485 A1;

(94) intermediate 2g: 2-cyclopropyloxazole-4-carbohydrazide, ethyl ester was made as described above;

(95) intermediate 2h: 2,5-dimethylthiazole-4-carbohydrazide, methyl ester precursor was previously synthesized using conventional esterification method (such as TMS-Cl in methanol) from commercially available acid;

(96) intermediate 2i: tert-butyl (4-(hydrazinecarbonyl)thiazol-2-yl)carbamate, ethyl 2-((tert-butoxycarbonyl)amino)thiazole-4-carboxylate precursor was previously Boc-protected using conventional method.

(97) intermediate 2j: 2-isopropylthiazole-4-carbohydrazide.

Synthesis of 4-methylthiazole-2-carbohydrazide 2k

(98) ##STR00203##

(99) In a 100 mL round-bottom flask equipped with a condenser, 4-methylthiazole-2-carboxylate 2.2k (500 mg, 2.92 mmol, 1 eq.) was dissolved in anhydrous EtOH (5 mL) and treated at RT with hydrazine monohydrate (216 L, 4.46 mmol, 1.5 eq.). The resulting solution was heated at reflux temperature for 18 hours. After allowing the reaction mixture to come to RT, the solution was concentrated under reduced pressure and the obtained crude was purified on a pad of silica (eluent: DCM/MeOH: 100/0 to 97/3) to afford 266 mg of 2k as a white solid (266 mg, 1.69 mmol, 57%). LCMS: P=90%, retention time=0.7 min, (M+H).sup.+: 158.

(100) The following intermediates were also prepared from the ad hoc carboxylic acids methyl or ethyl esters using General Method C:

(101) Intermediate 2l: 4,5-dimethylthiazole-2-carbohydrazide, prepared from ester 5.3. This latter was prepared in two steps from commercial thiazole 5.1 (procedure adapted from Castells, J. et al., Tetrahedron Lett., 1985, 26, 5457-5458).

(102) ##STR00204##

Step 1: Synthesis of 4,5-dimethylthiazole-2-carboxylic acid 2.1l

(103) A solution of 2.01 (3.0 g, 25.7 mmol, 1 eq.) in dry THF (50 mL) was degassed using vacuum pump and backfilled with N.sub.2 (repeated three times). The solution was then cooled to 78 C. and n-butyllithium (2.5M in hexanes, 11.3 mL, 28.3 mmol, 1.1 eq.) was added. The solution was stirred for 30 min at 78 C. and then the solution was placed under CO.sub.2 atmosphere (bubbling directly into the solution). After 1 hour of stirring at 78 C., the solution was allowed to warm to room temperature. HCl 1N (25 mL) and EtOAc (200 mL) were added. After separation of both phases, the aqueous phase was extracted with DCM (2100 mL). The organic phases were combined, washed with water, dried over MgsO.sub.4, filtered and concentrated under reduced pressure to afford acid 2.11 (3.0 g, 6.30 mmol) which was used in the next step without further purification.

Step 2: Synthesis of methyl 4,5-dimethylthiazole-2-carboxylate 2.2l

(104) To a solution of acid 2.11 (3.0 g, 6.30 mmol, 1 eq.) in commercial dry MeOH (12 mL) was added at RT chlorotrimethylsilane (4.0 mL, 31.5 mmol, 5 eq.) dropwise. The resulting solution was stirred at 60 C. for 14 hours. The reaction mixture was cooled down to RT, diluted with DCM (100 mL) and quenched with a saturated solution of NaHCO.sub.3 (50 mL). The aqueous phase was extracted with DCM (250 mL). The organic phases were combined, washed with brine (100 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude was purified by flash chromatography on silica gel (eluent Pet. Ether/EtOAc: 100/0 to 80/20) to afford 2.2l (1.32 g, 7.7 mmol, 55%). LCMS: P=33%, retention time=2.1 min, (M+H).sup.+: 172.

(105) Intermediate 2m: 3-methyl-1,2,4-oxadiazole-5-carbohydrazide, prepared from ester 2.2m using General Method C. This latter was prepared in one step from acetimidamide 2.0m (adapted from Street Leslie J. et al, J. Med. Chem., 2004, 47(14), 3642-3657).

(106) ##STR00205##

(107) To a solution of (E)-N-hydroxyacetimidamide 2.0m (1.0 g, 13.50 mmol, 1 eq.) and pyridine (4.35 mL, 54.0 mmol, 4 eq.) in dry DCM (40 mL) was added at RT ethyloxalyl chloride (2.4 g, 18.0 mmol, 1.3 eq.). The solution was stirred at reflux for 14 hours. The reaction mixture was cooled down to RT and quenched with NH.sub.4Cl sat. (30 mL). The aqueous phase was extracted with DCM (250 mL). The organic phases were combined, washed with NaHCO.sub.3 sat. (50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give 2.2m as yellow oil (1.32 g, 8.45 mmol, 63%) which was used in the next step without further purification. LCMS: P=92%, retention time=2.0 min, (M+H).sup.+: 157.

(108) Intermediate 2n: 3-methyl-1,2,4-thiadiazole-5-carbohydrazide, prepared from ester 2.2m using General Method C. This latter was prepared in one step from acetamide 2.0n, reagents 2.0n and 2.0n (adapted from U.S. Pat. No. 5,583,092A1).

(109) ##STR00206##

(110) A solution of 2.0n (500 mg, 8.46 mmol, 1 eq.), and 2.0n (820 L, 9.31 mmol, 1.2 eq.) in dry toluene (23 mL) was stirred at reflux for 2 hours. The solvent was then evaporated under reduced pressure and the residue was dissolved again in toluene (11.3 mL). 2.0n (2.0 mL, 25.4 mmol, 3 eq.) was added to the solution and the resulting mixture was stirred at reflux for 4 hours. The solvent was evaporated and the obtained crude was purified by flash chromatography on silica gel (eluent: DCM 100%) to obtain the desired ester 2.2n (150 mg, 0.95 mmol, 11%) as a brown oil. LCMS: P=97%, retention time=1.8 min, (M+H).sup.+: 159.

(111) Intermediate 2o: 4-methyloxazole-2-carbohydrazide. Prepared from ester 2.2o using General Method C. This latter was prepared in one step from 4-methyloxazole 2.0o.

(112) ##STR00207##

(113) To a solution of 2.0o (1.0 g, 12.0 mmol, 1 eq.) in commercially dry THF (50 mL) was added at 78 C. under Ar atmosphere n-BuLi (2.5M in hexanes, 5.30 mL, 13.24 mmol, 1.1 eq.). After 30 minutes of stirring at 78 C., ethylchloroformate (1.16 mL, 12.13 mmol, 1.0 eq.) was added dropwise. After 30 minutes of stirring, the dry ice bath was removed and the resulting solution was allowed to warm to RT and stirred for 14 hours. HCl 1N (15 mL) and EtOAc (30 mL) were added. After separation of both phases, the aqueous phase was extracted with DCM (210 mL). The organic phases were combined, washed with brine (20 mL), dried over MgsO.sub.4, filtered and concentrated under reduced pressure. The obtained crude was purified by flash chromatography on silica gel (eluent: DCM/MeOH: 100/0 to 99.5/0.5) to afford ester 2.2o (240 mg, 1.55 mmol, 13%) as a colorless oil. LCMS: P=96%, retention time=2.0 min, (M+H).sup.+: 156.

(114) Intermediate 2p: 3-isopropyl-1,2,4-thiadiazole-5-carbohydrazide, prepared from ester 2.2p using General Method C. This latter was prepared in one step from isobutyramide 2.0p, reagents 2.0p and 2.0p as depicted below.

(115) ##STR00208##

(116) A solution of 2.0p (500 mg, 5.74 mmol, 1 eq.), and 2.0p (555 L, 6.31 mmol, 1.2 eq.) in dry toluene (15 mL) was stirred at reflux for 2 hours. The solvent was then evaporated under reduced pressure and the residue was dissolved again in toluene (7.6 mL). 2.0p (900 L, 11.34 mmol, 2 eq.) was added to the solution and the resulting mixture was stirred at reflux for 4 hours. The solvent was evaporated and the obtained crude 2.2p (587 mg, 3.15 mmol, 55%) was used in the next step without further purification. LCMS: P=45%, retention time=2.3 min, (M+H).sup.+: 187.

(117) Intermediate 2q: 1,3-dimethyl-1H-pyrazole-5-carbohydrazide was prepared from commercial ethyl ester using General Method C.

Synthesis of 6-methylpicolinohydrazide 2r

(118) ##STR00209##

Step 1: Synthesis of methyl 6-methylpicolinate 2.2r

(119) To a solution of 6-methylpicolinic acid 2.1r (3 g, 21.88 mmol) in anhydrous MeOH (70 mL) at RT under N.sub.2 atmosphere was added TMS-C1 (13.88 mL, 109 mmol). The reaction mixture was left stirring at 60 C. overnight whereupon the mixture was concentrated under reduced pressure to afford 5.51 g of yellow oil used without further purification in next step. LCMS: P=95%, retention time=1.02 min, (M+H).sup.+: 152.

Step 2: Synthesis of 6-methylpicolinohydrazide 2r

(120) To a solution of crude methyl 6-methylpicolinate 2.2r (5.51 g, 21.88 mmol) in EtOH (22 mL) at RT was added hydrazine monohydrate (10.61 mL, 219 mmol). The reaction mixture was heated to reflux for 90 min. After allowing the reaction mixture to reach RT, the solution was concentrated under reduced pressure and purified by silica gel chromatography (eluent: DCM/MeOH: 100/0 to 96/4) to afford the desired product 2r as white solid (2.34 g, 15.48 mmol, 71%). LCMS: P=100%, retention time=0.54 min, (M+H).sup.+: 152.

(121) In one embodiment 2.5 to 20 equivalents of hydrazine hydrate was used to carry out this reaction using a temperature range from RT to reflux.

(122) In one embodiment, hydrazide 2 was recrystallized and/or precipitated. The following intermediates were also prepared from the ad hoc carboxylic acids or carboxylic acid ethyl ester using General Method C:

(123) intermediate 2s: 6-hydroxypicolinohydrazide,

(124) intermediate 2t: 6-bromopicolinohydrazide.

I.4. Step 3: Cyclodehydration Leading to Triazolopiperazine 3

Method D: Cyclodehydration and AcydolysisBoc Protection

(125) Method D is the procedure used for the synthesis of the triazolopiperazine 3 and is detailed below:

(126) ##STR00210##

(127) Method D is illustrated by the synthesis of intermediates 3a, 3f and 3g wherein the protecting group is Boc.

Synthesis of 2-methyl-4-(5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole hydrochloride 3a

(128) ##STR00211##

Step 1: Synthesis of tert-butyl 8-methyl-3-(2-methylthiazol-4-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate 3.1a

(129) In a 100 mL round-bottom flask equipped with a condenser, imino-ether 1b (1.089 g, 4.77 mmol, 1 eq.) was dissolved in commercial anhydrous EtOH (20 mL), to which was added 2-methylthiazole-4-carbohydrazide 2a (750 mg, 4.77 mmol, 1 eq.) in one portion. The resulting solution was stirred under reflux overnight. The reaction mixture was cooled down to RT and the solvent was removed under reduced pressure. The crude compound was then purified on silica gel (DCM/MeOH: 99/1 to 98/2) to afford the desired product 3.1a as white solid (1.07 g, 3.33 mmol, 70%). LCMS: P=100%, retention time=2.1 min, (M+H).sup.+: 321.

Step 2 Synthesis of 2-methyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazolehydrochloride 3a

(130) HCl 4M solution in 1,4-dioxane (8.32 mL, 33.3 mmol) was added in one portion to a solution of 3.1a (1.07 g, 3.33 mmol) in commercial iso-propanol (20 mL). The reaction mixture was stirred at 60 C. After 1.5 h (complete conversion by LC-MS), the reaction mixture was allowed to cool to room temperature and then further cooled to 0 C. with an ice bath. Thereupon, 10 mL of Et.sub.2O was added. After 15 min stirring, the precipitate was filtered and dried in vacuo to afford 3a as white solid. Yield: 736 mg (86%). LCMS: P=97%, retention time=0.5 min, (M+H).sup.+: 222.

(131) The following intermediates were also prepared from the ad hoc reagents and intermediates using General Method D:

(132) intermediate 3b: 4-(5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-(trifluoromethyl)thiazole hydrochloride, from intermediates la and 2b;

(133) intermediate 3c: 4-(5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-vinylthiazole, from intermediates 1a and 2d then the Boc aminoethyl derivative obtained 3.1c was deprotected in acidic conditions (as step 2 above using only 2 eq of HCl in dioxane) followed by dimethylamine elimination (using 10 eq of NaH and MeI at RT), then vinyl derivative 3.1c obtained was subjected to step 2 above to afford 3c;

(134) intermediate 3d: 4-(5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-isopropyloxazole hydrochloride, from intermediates 1a and 2f;

(135) intermediate 3e: 2-isopropyl-4-(5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole hydrochloride, from intermediates 1a and 2j.

Synthesis of 4-methyl-2-(5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole 3f

(136) ##STR00212##

Step 1: Synthesis of tert-butyl 3-(4-methylthiazol-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate 3.1f

(137) Imino-ether 1a (148 mg, 0.649 mmol, 1 eq.) was dissolved in anhydrous EtOH (3 mL) at RT, to which was added 2-methylthiazole-4-carbohydrazide 2k(102 mg, 0.649 mmol, 1 eq.). The resulting solution was stirred under reflux overnight. The reaction mixture was cooled to RT and the solvent was removed under reduced pressure. The crude compound was then purified on silica gel (DCM/MeOH: 99/1 to 98/2) to afford the desired product 3.1f as yellow solid (174 mg, 83%). LCMS: P=93%, retention time=2.2 min, (M+H).sup.+: 322.

Step 2 Synthesis of 2-methyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazolehydrochloride 3f

(138) 4M HCl in dioxane (2.71 mL, 10.83 mmol) was added to a solution of Boc-triazolopiperazine 3.1f (1.07 g, 3.33 mmol) in iso-propanol (3 mL) at RT. The reaction mixture was stirred at 60 C. After 1.5 h (complete conversion by LC-MS), the reaction mixture was allowed to cool to room temperature and then further cooled to 0 C. with an ice bath. Thereupon, 5 mL of Et.sub.2O was added. After 30 min stirring, the precipitate was filtered off and dried in vacuo to afford 3f as a white solid (132 mg, 95%). LCMS: P=97%, retention time=0.9 min, (M+H).sup.+: 222.

Synthesis of 8-methyl-3-(6-methylpyridin-2-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-n]pyrazine 3g

(139) ##STR00213##

Step 1: Synthesis of tert-butyl 8-methyl-3-(6-methylpyridin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-carboxylate 3.1g

(140) Iminoether 1b (468 mg, 1.93 mmol, 1 eq.) was dissolved in anhydrous EtOH (2 mL), to which was added carbohydrazide 2r (270 mg, 1.79 mmol, 1 eq.). The resulting mixture was stirred at 135 C. in oil bath for 63 h. The reaction mixture was allowed to reach RT whereupon volatiles were removed under reduced pressure. The crude compound was then purified using silica gel chromatography (DCM/MeOH: 99/1 to 98/2) to afford the desired product 3.1g as yellow oil (380 mg, 1.15 mmol, 65%). LCMS: P=95%, retention time=2.2 min, (M+H).sup.+: 330.

Step 2: Synthesis of 8-methyl-3-(6-methylpyridin-2-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine 3g dihydrochloride salt

(141) 4M HCl in dioxane (5.77 mL, 23.07 mmol) was added to a solution of 3.1g (380 mg, 1.15 mmol) in iso-propanol (10 mL). The reaction mixture was stirred at 60 C. for 1 h. The reaction mixture was allowed reach RT and then further cooled to 0 C. The precipitate eventually obtained was filtered and dried in vacuo to afford 3g as yellow solid. (367 mg, quant.). LCMS: P=92%, retention time=0.2 min, (M+H).sup.+: 230.

(142) The following intermediates were also prepared from the ad hoc reagents and intermediates using General Method D:

(143) intermediate 3h: 6-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)pyridin-2-ol hydrochloride salt, from intermediates 1c and 2s;

(144) intermediate 3i: 3-(6-bromopyridin-2-yl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine dihydrochloride salt, from intermediates 1b and 2t.

Method E: Cyclodehydration and AcydolysisDMB Protection

(145) Method E is the procedure used for the synthesis of the triazolopiperazine 3 and is detailed below:

(146) ##STR00214##

(147) Method E is illustrated by the synthesis of intermediates 3j and 3q wherein the protecting group is DMB.

Synthesis of 2-ethyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole 3j

(148) ##STR00215##

Step 1: Synthesis of 4-(7-(2,4-dimethoxybenzyl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-ethylthiazole 3.1j

(149) In a 10 mL round-bottom flask equipped with a condenser, imino-ether 1c (790 mg, 2.70 mmol, 1 eq.) was dissolved in anhydrous EtOH (2.5 mL), to which was added 2-methylthiazole-4-carbohydrazide 2c (462 mg, 2.70 mmol, 1 eq.) in one portion. The resulting solution was stirred at 135 C. overnight. Thereafter, the reaction mixture was brought to RT and the volatiles removed under reduced pressure. The crude compound was then purified using silica gel chromatography (DCM/MeOH: 99/1 to 98/2) to afford the desired product 3.1j as yellow solid (837 mg, 2.10 mmol, 78%). LCMS: P=97%, retention time=1.9 min, (M+H).sup.+: 400.

Step 2 Synthesis of 2-ethyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole 3j

(150) In a round-bottom flask containing 10 ml DCM was added 4-(7-(2,4-dimethoxybenzyl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-ethylthiazole 3.1j (0.837 g, 2.10 mmol). Then, TFA (10.48 mL, 141 mmol), was added to the reaction mixture at RT. After 30 min stirring, the mixture was concentrated. Then ca 25 mL DCM was added to the residue thus obtained, and washed with saturated NaHCO.sub.3 (15 mL). The aqueous layer was extracted twice with 25 mL of DCM, the organic layers were washed with 25 mL of brine, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to obtain crude 3e as a pink oil (500 mg, 96%). The crude 3j was directly used in the next step without further purification.

(151) In one embodiment, alternative work-up equally used involved treatment of the dried residue obtained above with 4 M HCl/dioxane (20 eq.) at RT under stirring. After 5 min, Et.sub.2O was added to help precipitation. This precipitate was filtered off under vacuum, washed with Et.sub.2O and dried under high vacuum to furnish 3j.

(152) The following intermediates were also prepared from the ad hoc reagents and intermediates using General Method E:

(153) intermediate 3k: 4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-vinylthiazole from intermediates 1c and 2d; the Boc aminoethyl derivative 3.1k isolated after condensation was first Boc-deprotected (8 eq of HCl/dioxane). Following dimethylamine elimination (using 10 eq of NaH and MeI at RT), the vinyl moiety obtained was then DMB-deprotected as in step 2 above to furnish 3k;

(154) intermediate 3l: 2-methyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)oxazole, from intermediates 1c and 2e;

(155) intermediate 3m 2-isopropyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)oxazole, from intermediates 1c and 2f;

(156) intermediate 3n: 2-cyclopropyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)oxazole, from intermediates 1c and 2g;

(157) intermediate 3o: 2,5-dimethyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole, from intermediates 1c and 2h;

(158) intermediate 3p: 4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazol-2-amine, from intermediates 1c and 2g.

Synthesis of 4,5-dimethyl-2-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole hydrochloride 3q

(159) ##STR00216##

Step 1: Synthesis of 2-(7-(2,4-dimethoxybenzyl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-4,5-dimethylthiazole 3.1q

(160) Iminoether 1d (768 mg, 2.63 mmol, 1 eq.) was dissolved in anhydrous EtOH (5 mL), to which was added 4,5-dimethylthiazole-2-carbohydrazide 21(450 mg, 2.63 mmol, 1 eq.) and the resultant reaction mixture was refluxed for 48 hours. The reaction mixture was then brought to RT and the volatiles was removed under reduced pressure, whereupon the isolated crude was purified using silica gel chromatography (DCM/MeOH: 100/0 to 98/2) to afford the desired product 3.1q (786 mg, 1.93 mmol, 74%). LCMS: P=65%, retention time=1.9 min, (M+H).sup.+: 400.

Step 2: Synthesis of 4,5-dimethyl-2-(8-methyl-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole 3c

(161) To 3.1q (0.786 g, 1.97 mmol) in anhydrous DCM (6.6 mL) at RT was added TFA (9.1 mL, 148 mmol) and the mixture refluxed for 30 min whereupon the volatiles were removed under vacuum. 4M HCl in dioxane (5 mL, 20 mmol) was added dropwise at RT with stirring. After 5 min, Et.sub.2O was added to help precipitation of the product, whereupon it was filtered, washed with Et.sub.2O and dried under vacuum to afford 3q (729 mg, 100%). LCMS: P=100%, retention time=1.6 min, (M+H).sup.+: 250.

(162) In one embodiment 20 eq. of TFA at RT in DCM (1:1 mixture DCM/TFA v/v) was used to carry out this reaction.

(163) The following intermediates were also prepared from the ad hoc reagents and intermediates using General Method E:

(164) intermediate 3r: 3-methyl-5-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-1,2,4-oxadiazole hydrochloride, from intermediates 1c and 2m;

(165) intermediate 3s: 3-methyl-5-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-1,2,4-thiadiazole hydrochloride, from intermediates 1c and 2n;

(166) intermediate 3t: 4-methyl-2-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)oxazole hydrochloride, from intermediates 1c and 2o;

(167) intermediate 3u: 3-isopropyl-5-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-1,2,4-thiadiazole hydrochloride, from intermediates 1c and 2p.

Method F: Cyclodehydration and AcydolysisPMB Protection

(168) Method F is the procedure used for the synthesis of the triazolopiperazine 3 and is detailed below:

(169) ##STR00217##

(170) Method F is illustrated by the synthesis of intermediate 3v wherein the protecting group is PMB.

Synthesis of 4-methyl-2-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole 3v

(171) ##STR00218##

Step 1: Synthesis of 2-(7-(4-methoxybenzyl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-4-methylthiazole 3.1v

(172) Imino-ether 1d (444 mg, 1.69 mmol, 1 eq.) was dissolved in anhydrous EtOH (5 mL), to which was added 2-methylthiazole-4-carbohydrazide 2k (266 mg, 1.69 mmol, 1 eq.) and the resultant solution was refluxed for 24 h. The reaction mixture was cooled to RT and the solvent was removed under reduced pressure. The crude compound was then purified on silica gel (DCM/MeOH: 99/1 to 98/2) to afford the desired product 3.1v as a pale yellow solid (383 mg, 1.07 mmol, 64%). LCMS: P=75%, retention time=1.9 min, (M+H).sup.+: 356.

Step 2: Synthesis of 4-methyl-2-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole 3v

(173) Anhydrous DCM (2.5 mL) was added at RT to 2-(7-(4-methoxybenzyl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-4-methylthiazole 3.1v (443 mg, 1.246 mmol, 1 eq.). TFA (2.5 mL, 33.5 mmol, 27 eq.) was then added and the reaction mixture refluxed for 15 h. The reaction was quenched by addition of NaHCO.sub.3 sat. solution. The layers were separated and the aqueous layer was basified to pH14 with NaOH 1M solution and was extracted with DCM (370 mL). Combined organic layers were washed with brine (70 mL), dried over MgSO4, filtered and concentrated under reduced pressure to afford 3v after vacuum during for 3 h without mass variation. (342 mg, 100%). LCMS: P=100%, retention time=1.2 min, (M+H).sup.+: 236.

(174) The following intermediate was also prepared from the ad hoc reagents and intermediates using General Method F:

(175) Intermediate 3w: 3-(1,3-dimethyl-1H-pyrazol-5-yl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine, from intermediates 1d and 2q.

I.5. Step 4: Acylation Leading to Final Products

Method G: Acylation and Chiral HPLC Purification

(176) Method G is the procedure used for the synthesis of the racemic product 4 and its purification to obtain final compounds n.sup.o X of general Formula I. Method G is detailed below:

(177) ##STR00219##

(178) Method G is illustrated by the synthesis of compounds n.sup.o 5, 19, 29 and 33 of general Formula I.

Synthesis of (3-(2-ethylthiazol-4-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(thiophen-2-yl)phenyl)methanone 4a and (R)-(3-(2-ethylthiazol-4-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(thiophen-2-yl)phenyl)methanone compound n.SUP.o .5

(179) ##STR00220##

(180) To a solution of crude 3j (250 mg, 1.003 mmol, 1 eq.) in anhydrous DCM (10 mL) were added, at RT, 4-(thiophen-2-yl)benzoyl chloride 4.1a(290 mg, 1.303 mmol, 1.3 eq.), followed by N-methylmorpholine (0.359 mL, 3.51 mmol, 3.5 eq.) dropwise over 15 sec. The reaction mixture was stirred at RT for 10 minutes and the milky suspension was poured into 10 mL of 1 M HCl solution. The aqueous phase was extracted with DCM (310 mL). The organic phases were combined, washed with 1 M NaOH (20 mL), brine (20 mL), dried over MgSO.sub.4 and evaporated to dryness. The residue was solubilized in DCM (4 mL) and Et.sub.2O was slowly added (5 mL) to induce precipitation. The solid was filtered off, washed with 2 mL of Et.sub.2O and dried under vacuum to afford 4a as yellow powder (234 mg, 0.537 mmol, 54%). LCMS: P=97%, retention time=2.4 min, (M+H).sup.+: 436.

(181) 4a was purified by chiral preparative HPLC according to the abovementioned method to yield title compound n.sup.o 5 as a white powder. LCMS: P=100%, retention time=4.3 min, (M+H).sup.+: 436; Chiral HPLC retention time: 14.0 min; ee >99%; .sup.1H-NMR (CDCl.sub.3): 8.02 (s, 1H), 7.70 (d, J=8.2, 2H), 7.47 (d, J=8.2, 2H), 7.31 (m, 2H), 7.12 (m, 1H), 5.77 (br, 1H), 4.83 (m, 1H), 4.63 (br, 1H), 4.26 (m, 1H), 3.53 (m, 1H), 3.07 (d, J=7.5, 2H), 1.74 (d, J=6.9, 3H), 1.43 (t, J=7.5, 3H).

Synthesis of (8-methyl-3-(4-methylthiazol-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(thiophen-2-yl)phenyl)methanone 4b and (R)-(8-methyl-3-(4-methylthiazol-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(thiophen-2-yl)phenyl)methanone compound n.SUP.o .19

(182) ##STR00221##

(183) To a solution of 3v (342 mg, 1.25 mmol, 1 eq.) in commercial anhydrous DCM (12 mL) at RT were added 4-(thiophen-2-yl)benzoyl chloride 4.1a(326 mg, 1.464 mmol, 1.17 eq.), followed by N-methylmorpholine (0.128 mL, 1.25 mmol, 1.0 eq.) dropwise over 15 sec. The reaction mixture was stirred at RT for 15 minutes and then diluted with DCM (60 mL). The organic layer was washed with water (40 mL), brine (50 mL), dried over MgSO.sub.4, filtered and evaporated under reduced pressure. The residue was purified on silica gel (DCM/MeOH: 98/2) to afford 4b as yellow oil with 88% purity by LCMS. Diethylether (10 mL) was added on obtained oil and mixture was sonicated. A white solid precipitated and was filtered. The filtrate was concentrated under reduced pressure and diethylether (5 mL) was added on the residue. After sonication, a second white precipitate was filtered. Both precipitates were merged to afford 4b as white solid (189 mg, 36%). LCMS: P=99%, retention time=4.4 min, (M+H).sup.+: 422.

(184) 4b was purified by chiral preparative HPLC according to the abovementioned method to yield title compound n.sup.o 19 as white powder. LCMS: P=100%, retention time=4.3 min, (M+H).sup.+: 422; Chiral HPLC retention time: 6.6 min, ee=94%; .sup.1H-NMR (CDCl.sub.3): 7.70 (d, J=8.2, 2H), 7.48 (d, J=8.2, 2H), 7.40-7.35 (m, 2H), 7.13-7.11 (m, 1H), 7.00 (m, 1H), 5.81 (br, 1H), 4.95 (dd, J.sub.1=3.3, J.sub.2=14.0, 1H), 4.60 (br, 1H), 4.27 (td, J.sub.1=3.9, J.sub.2=12.7, 1H), 3.51 (m, 1H), 2.50 (s, 3H), 1.75 (d, .sub.J=6.9, 3H).

(185) When hydrochloride salt of 3 was used, 2.2 eq. of N-methylmorpholine were added.

Synthesis of Compound N.SUP.o .29: (R)-[1,1-biphenyl]-4-yl(8-methyl-3-(6-methylpyridin-2-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone

(186) ##STR00222##

(187) To a solution of 3g (500 mg, 1.65 mmol, 1 eq.) in anhydrous DCM (10 mL) were added at RT [1,1-biphenyl]-4-carbonyl chloride 4.1c (430 mg, 1.98 mmol, 1.2 eq.), followed by N-methylmorpholine (507 L, 4.96 mmol, 3.00 eq.). The reaction mixture was stirred at RT for 30 min. whereupon saturated NaHCO.sub.3 solution (10 mL) and DCM (5 mL) were added to the reaction mixture. The organic phase was extracted, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The crude product was purified using silica gel chromatography (eluent: DCM/MeOH: 98:2) to afford 268 mg of 4c. LCMS: P=98%, retention time=4.2 min, (M+H).sup.+: 410.

(188) 4c was purified by chiral preparative HPLC according to the abovementioned method to yield title compound n.sup.o 29 as a white powder. LCMS: P=100%, retention time=4.2 min, (M+H).sup.+: 410; Chiral HPLC retention time: 4.7 min; ee >99%. .sup.1H-NMR (CDCl.sub.3): 8.11 (d, J=7.7, 1H), 7.67-7.40 (m, 10H), 7.20 (d, J=6.7, 1H), 5.78 (bs, 1H), 5.00 (dd, J.sub.1=3.3, J.sub.2=14.0, 1H), 4.67 (br, 1H), 4.37 (m, 1H), 3.51 (m, 1H), 2.58 (s, 3H), 1.76 (d, J=6.9, 3H).

(189) The procedure used for the synthesis of compound n.sup.o 33 is the following:

(190) ##STR00223##

Step 1: Synthesis of (3-(6-bromopyridin-2-yl)-8-methyl-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(thiophen-2-yl)phenyl)methanone 4d

(191) 4d was prepared from 3i and 4.1a according to General Method G.

Step 2: Synthesis of 6-(8-methyl-7-(4-(thiophen-2-yl)benzoyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)picolinonitrile 4e

(192) A mixture of 4d (140 mg, 0.291 mmol) and zinc cyanide (137 mg, 1.166 mmol) in DMA (2 mL) at RT was degassed. Then Pd(PPh.sub.3).sub.4 (67.4 mg, 0.058 mmol) was added (67.4 mg, 0.058 mmol). The reaction mixture was stirred at 115 C. for 30 min. whereupon DCM (30 mL) was added and the organic layer extract was washed with water (230 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure.

(193) The residue was purified using silica gel chromatography (DCM/MeOH: 100/0 to 98/2) to afford 4d as white solid (8 mg, 6%). LCMS: P=90%, rt=4.2 min, (M+H).sup.+: 427.

(194) 4d was purified by chiral preparative HPLC according to the abovementioned method to yield title compound n.sup.o 33 as white powder. LCMS: P=100%, retention time=4.2 min, (M+H).sup.+: 427; Chiral HPLC retention time: 18.8 min; ee=98%.

II. Chiral Synthesis

II.1. General Synthetic Scheme for Chiral Synthesis

(195) Compounds of the invention were synthesized using the chiral process of the invention described in Scheme 30.

(196) ##STR00224##

(197) Chiral ketopiperazine A was protected with a DMB group and converted to iminoether D by using the Meerwein reagent (Et.sub.3OBF.sub.4). Condensation reaction between the acyl hydrazide E and iminoether D was conducted under heating conditions in ethanol to provide DMB protected piperazine F that was subsequently deprotected with HCl in dioxane to yield compound of Formula II.

(198) In one embodiment, the DMB deprotection step (from F to II) was carried out using TFA in DCM.

(199) In one embodiment, the DMB group deprotection step (from F to II) is carried out using

(200) TFA in DCM at RT, followed by either TFA salt exchange with HCl or extraction at high pH recovering free piperazine II.

(201) Acylation with the appropriate acid chloride afforded the final product of Formula I typically in >90% enantiomeric excess (chiral HPLC).

General Method H

(202) General Method A is the procedure used for the synthesis of (R)-4-(2,4-dimethoxybenzyl)-3-methylpiperazin-2-one (R)-C (cf. Scheme 30).

(203) In a round-bottom flask, were sequentially introduced (R)-3-methylpiperazin-2-one (R)-A (725 mg, 6.35 mmol, 1 eq.), 2,4-dimethoxybenzaldehyde B (1.16 g, 6.99 mmol, 1.1 eq.), acetic acid (545 l, 9.53 mmol, 1.5 eq.) and sodium triacetoxyborohydride (1.88 g, 8.89 mmol, 1.4 eq.) in commercial anhydrous acetonitrile (65 mL), at RT, under N.sub.2 atmosphere. The reaction was stirred at RT overnight. The reaction mixture was quenched carefully at 0 C. with saturated NaHCO.sub.3 solution (100 mL) until no more bubbling was observed. Aqueous and organic layers were separated. The aqueous layer was extracted with EtOAc (3100 mL) and the combined organic layers were washed with brine, dried over MgSO.sub.4 filtered, and concentrated under reduced pressure to afford the title compound as yellow oil. The crude compound was then purified on silica gel (DCM/MeOH: 98/2 to 95/5) to afford the desired product (R)-C as a viscous pale yellow oil. Yield: 1.65 g, 98%. LCMS: P=100%, retention time=1.6 min, (M+H).sup.+: 265; chiral HPLC retention time=41.5 min, ee >99%; .sup.1H-NMR (CDCl.sub.3): 7.23 (d, J=8.9, 1H), 6.49 (d, J=8.9, 1H), 6.46 (s, 1H), 6.29 (br, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.78 (d, J.sub.AB=15.0, 1H), 3.49 (d, J.sub.AB=15.0, 1H), 3.27 (m, 2H), 3.19 (m, 1H), 2.95 (m, 1H), 2.48 (m, 1H), 1.48 (d, J=6.8, 3H).

(204) The (S)-4-(2,4-dimethoxybenzyl)-3-methylpiperazin-2-one (S)-C was also prepared using General Method H starting from (S)-3-methylpiperazin-2-one (S)-A. Yield: 300 mg, 99%. LCMS: P=100%, retention time=1.6 min, (M+H).sup.+: 265; chiral HPLC retention time=26.6 min, ee >99%.

General Method I

(205) General Method I is the procedure used for the synthesis of (R)-1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine (R)-D (cf. Scheme 30) as detailed below.

(206) Oven dried (115 C.) sodium carbonate (2.48 g, 23.40 mmol, 2.25 eq.) was placed in a round-bottom flask. The round-bottom flask was backfilled with Ar and then capped with a rubber septum. A solution of (R)-4-(2,4-dimethoxybenzyl)-3-methylpiperazin-2-one (R)-C (2.75 g, 10.40 mmol, 1 eq.) in anhydrous DCM (35 mL) was added, followed by freshly prepared triethyloxonium tetrafluoroborate (2.48 g, 13.05 mmol, 1.25 eq.) in one portion. Thereafter the reaction mixture was stirred further at RT for 1 hour, whereupon the reaction mixture was diluted with saturated aqueous NaHCO.sub.3 (100 mL). The aqueous layer was extracted with DCM (3200 mL). The organic layers were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford 3.1 g of yellow oil. The crude compound was then purified on silica gel (EtOAc/MeOH: 99/1) to afford the desired product (R)-D as a pale yellow oil. Yield: 1.44 g, 48%. LCMS: P=95%, retention time=1.8 min, (M+H2O+H).sup.+: 311; chiral HPLC retention time=12.3 min, ee >97%. .sup.1H-NMR (CDCl.sub.3): 7.23 (d, J=8.8, 1H), 6.48 (d, J=8.8, 1H), 6.44 (s, 1H), 4.02 (m, 2H), 3.92 (s, 6H), 3.86 (d, J.sub.AB=14.0, 1H), 3.46 (d, J.sub.AB=14.0, 1H), 3.44 (m, 2H), 3.10 (m, 1H), 2.79 (m, 1H), 2.32 (m, 1H), 1.35 (d, J=6.8, 3H), 1.24 (t, J=6.0, 3H).

(207) The (S)-1-(2,4-dimethoxybenzyl)-5-ethoxy-6-methyl-1,2,3,6-tetrahydropyrazine(S)-D was also prepared using General Method I starting from (S)-C (46 mg, 0.16 mmol, 59%). LCMS: P=100%, retention time=1.8 min, (M+H2O+H).sup.+: 311; chiral HPLC retention time=11.3 min, ee=96%.

General Method J

(208) General Method J is the procedure used for the synthesis of hydrazide E.a (cf scheme 31) as detailed below.

(209) ##STR00225##

Synthesis of 2-methylthiazole-4-carbohydrazide E.a

(210) In a 100 mL round-bottom flask equipped with a condenser, ethyl 2-methylthiazole-4-carboxylate E.1a (10 g, 58.4 mmol, 1 eq.) was dissolved in anhydrous EtOH (25 mL) and treated at RT with hydrazine monohydrate (17.0 mL, 354.4 mmol, 6 eq.). The resulting yellow solution was heated at reflux temperature for 14 h. After allowing the reaction mixture to come to RT, the solution was concentrated under reduced pressure to afford 13.4 g of a brown oil. Co-evaporations using 3200 mL of a mixture of commercial anhydrous DCM:MeOH (1:1) were performed to remove residual water. The residue was then recrystallized from hot EtOH (60 mL): after total dissolution, the mixture was then allowed to cool down to RT and then put at 0 C. (with an ice bath) for 40 min. The obtained crystals were filtered and washed with cooled (0 C.) EtOH (230 mL). The orange solid was dried under vacuum for 1 h to afford E.a (5.85 g, 37.2 mmol, 64%). LCMS: P=100%, retention time=0.5 min, (M+H).sup.+: 158; .sup.1H-NMR (CDCl.sub.3): 8.32 (br, 1H), 7.96 (s, 1H), 4.07 (br, 2H), 2.70 (s, 3H).

General Method K

(211) General Method K is the general procedure used for the synthesis of chiral triazolopiperazine intermediates F (cf. scheme 30) and is detailed below in scheme 32 with the synthesis of (R)-4-(7-(2,4-dimethoxybenzyl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-methylthiazole(R)-F.a.

(212) ##STR00226##

(213) In a 50 mL round-bottom flask equipped with a condenser, imino-ether (R)-D (4.51 g, 14.96 mmol, 1 eq.) was dissolved in anhydrous EtOH (15 mL), to which was added 2-methylthiazole-4-carbohydrazide E.a (2.35 g, 14.96 mmol, 1 eq.) in one portion. The resulting solution was stirred at 70 C. for 6 hours. The reaction mixture was cooled down to RT and the solvent was removed under reduced pressure. The crude compound was then purified by silica gel chromatography (DCM/MeOH: 99/1 to 95/5) to afford the desired product (R)-F.a as pale yellow foamy solid. Yield: 3.78 g, 65%. LCMS: P=96%, retention time=1.8 min, (M+H).sup.+: 386; chiral HPLC retention time=13.9 min, ee=95%; .sup.1H-NMR (CDCl.sub.3): 7.85 (s, 1H), 7.19 (s, 1H), 6.41 (m, 2H), 4.38 (m, 1H), 4.16 (m, 1H), 3.96 (m, 1H), 3.86 (d, J.sub.AB=15.0, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.56 (d, J.sub.AB=15.0, 1H), 3.11 (m, 1H), 2.66 (s, 3H), 2.62 (m, 1H), 1.64 (d, J=6.6, 3H); .sup.13C-NMR (CDCl.sub.3): 166.2, 160.2, 158.8, 154.6, 148.1, 143.1, 130.9, 118.8, 118.2, 104.2, 98.5, 77.6, 77.2, 76.8, 70.4, 70.2, 55.4, 55.4, 55.8, 50.2, 45.8, 44.2, 19.2, 17.7, 15.7.

(214) In a round-bottom flask equipped with a condenser, imino-ether (R)-D(890 mg, 3.04 mmol, 1 eq.) was dissolved in anhydrous EtOH (3 mL), to which was added 2-methylthiazole-4-carbohydrazide E.a (479 mg, 3.04 mmol, 1 eq.). The resulting solution was stirred at 70 C. for 7 hours, then brought to RT and the volatiles removed under reduced pressure. The crude compound was then purified by silica gel chromatography (DCM/MeOH: 99/1 to 95/5) to afford the desired product F.a as pale yellow oil. Yield: 685 mg, 58%. LCMS: P=96%, retention time=1.8 min, (M+H).sup.+: 386; chiral HPLC retention time: 14.3 min, ee=95%; .sup.1H-NMR (CDCl.sub.3): 7.85 (s, 1H), 7.19 (s, 1H), 6.41 (m, 2H), 4.38 (m, 1H), 4.16 (m, 1H), 3.96 (m, 1H), 3.86 (d, J.sub.AB=15.0, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 3.56 (d, J.sub.AB=15.0, 1H), 3.11 (m, 1H), 2.66 (s, 3H), 2.62 (m, 1H), 1.64 (d, J=6.6, 3H).

(215) The (S)-4-(7-(2,4-dimethoxybenzyl)-8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)-2-methylthiazole(S)-F.a was also prepared using General Method K starting from (S)-D(36 mg, 0.09 mmol, 54%). LCMS: P=90%, retention time=1.8 min, (M+H).sup.+: 386; chiral HPLC retention time=21.0 min, ee=94.0%.

General Method L

(216) General Method E is the general procedure used for the synthesis of compounds of Formula II salts (cf. compounds II in scheme 30) and is detailed below in scheme 33 with the synthesis of compound no 11-1: (R)-8-methyl-3-(2-methylthiazol-4-yl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-7-ium chloride(R)-II-1.

(217) ##STR00227##

(218) In a 50 mL round-bottom flask equipped with a condenser, were introduced (R)-F.a (262 mg, 0.68 mmol, 1 eq.) followed by a solution of HCl 4 M in dioxane (3.4 mL, 13.60 mmol, 20 eq.) in one portion. The resulting yellow solution was stirred at 100 C. After 6 hours, i-PrOH (6 mL) was added to the hot reaction mixture. The solution was then allowed to reach RT by removing the oil bath. Et.sub.2O (15 mL) was then added and the obtained precipitate was filtered off, washed with Et.sub.2O (3 mL) and air-dried overnight to afford (R)-II-1 (235 mg, 0.86 mmol, 100%) as a pink solid which was used in the next step without further purification.

(219) The (S)-2-methyl-4-(8-methyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)thiazole(S)-II-1 was prepared using TFA procedure, starting from (S)-F.a as followed:

(220) (S)-F.a (36 mg, 0.09 mmol, 1 eq.) was dissolved in dry DCM (500 L). TFA (467 L, 6.0 mmol, 65 eq.) was added dropwise at RT. After 30 minutes, the dark pink reaction mixture was quenched carefully with a saturated solution of NaHCO.sub.3 (10 mL). The aqueous phase was extracted with DCM (310 mL). Organic phases were combined, washed with brine (10 mL), dried over MgSO4, filtered and concentrated under reduced pressure to afford the free amine (S)-II-1 as white solid (43 mg, 0.183 mmol, 100%) which was used in the next step without further purification.

(221) Determination of Enantiomeric Excess:

(222) As aforementioned, given that chiral LC determination of % ee proved difficult for compounds of Formula II, specifically due to technical chiral LC issues in dealing with such amines, the % ee was determined through the product formed at the subsequent step wherein the amine was acylated to furnish the final products exemplified through but not limited to compound n.sup.o 1 of Formula I.

General Method M

(223) General Method M is the general procedure used for the synthesis of chiral triazolopiperazine compounds of the invention and is detailed below with the synthesis of (R)-(8-methyl-3-(2-methylthiazol-4-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(thiophen-2-yl)phenyl)methanone (R)-compound n.sup.o 1 of Formula I (hereunder noted I-1).

(224) ##STR00228##

(225) To a solution of crude (R)-II-1 (235 mg, 0.67 mmol, 1 eq.) in anhydrous DCM (10 mL) were added at 0 C. 4-(thiophen-2-yl)benzoyl chloride 4.1a(165 mg, 0.742 mmol, 1.3 eq.), followed by N-methylmorpholine (163 L, 1.48 mmol, 2.2 eq.) dropwise over 15 sec. The reaction mixture was stirred at RT for 10 minutes and, the milky suspension was poured into 10 mL of 1 M HCl. The aqueous phase was extracted with DCM (310 mL). The organic phases were combined, washed with 1 M NaOH (20 mL), brine (20 mL), dried over MgSO.sub.4 and evaporated to dryness. The crude compound was purified by silica gel chromatography (eluent: EtOAc/MeOH: 98/2) to afford the desired product (R)-I-1 as a white foam. Yield: 158 mg, 55%. LCMS: P=97%, retention time=4.0 min, (M+H).sup.+: 422; Chiral HPLC retention time=15.4 min, ee=95%; .sup.1H-NMR (CDCl.sub.3): 7.93 (s, 1H), 7.61 (d, J=7.9, 2H), 7.40 (d, J=7.9, 2H), 7.31 (m, 2H), 7.04 (m, 1H), 5.73 (m, 1H), 4.78 (m, 1H), 4.46 (m, 1H), 4.14 (m, 1H), 3.47 (m, 1H), 2.70 (s, 3H), 1.68 (d, J=6.7, 3H). .sup.13C-NMR (CDCl3): 170.3, 166.5, 151.9, 148.0, 142.4, 136.4, 128.1, 125.8, 124.0, 119.3, 77.4, 77.0, 76.6, 44.8, 30.7, 19.6, 19.1.

(226) Identical % ee was obtained for compounds (R)-I-1 and (R)-F.a thus confirming that no detectable racemization occurs during the acidolytic deprotection and N-acylation steps.

(227) Compound (S)-(8-methyl-3-(2-methylthiazol-4-yl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)(4-(thiophen-2-yl)phenyl)methanone (S)-I-1 was also prepared using General Method F starting from (S)-II-1(16 mg, 38.0 mol, 40%). LCMS: P=90%, retention time=4.0 min, (M+H).sup.+: 386.1; chiral HPLC retention time=11.0 min, ee=92%.

(228) X-Ray Crystallographic Characterization of Compound (R)-I-1.

(229) Compound(R)-I-1 was characterized by single crystal X-ray spectroscopy thus establishing the configuration of the more active enantiomer as being the (R)-configuration (see FIG. 1).

(230) Method.

(231) All data were recorded on a MAR345 image plate (MARRESEARCH) using MoK radiation (=0.71073). X-rays were generated on a RIGAKU rotating anode generator with power settings of 50 KV and 70 mA. A Zr filter is used to eliminate the MoK radiation. A suitable crystal was chosen under a microscope, mounted in a nylon loop and aligned on the goniometer prior to the x-ray experiment. A total of 174 images corresponding to a 2.0 phi rotation were collected at room temperature. The reflections on the diffraction images were indexed and integrated using the Automar data processing suite (MARRESEARCH). During the integration the friedel pairs were kept unmerged in order to preserve the anomalous signal needed for absolute structure determination. Xprep (Bruker) was used to determine the spacegroup and to generate the reflection and instruction files for structure determination and subsequent refinement. Structure solution was performed by SHELXS and the refinement was done by SHELXL (A short history of SHELX. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122). The free rotation around the C6-C1 (C1A or C1B) results in rotational isomerism in a 58/42% ratio as seen in FIG. 1 below. As depicted in the X-ray Figure below the chirality of C22 carbon atom is established as R. (H. D. Flack (1983). On Enantiomorph-Polarity Estimation. Acta Cryst A39: 876-881; J. Appl. Cryst. (2008), 41, 96-103.)

(232) X-Ray Crystallographic Characterization of Compound (S)-I-1.

(233) Compound(S)-I-1 was characterized by single crystal X-ray spectroscopy thus establishing the configuration of the more active enantiomer as being the (R)-configuration (see FIG. 2).

(234) Method.

(235) All data were recorded on a MAR345 image plate (MARRESEARCH) using MoK radiation (=0.71073). X-rays were generated on a RIGAKU rotating anode generator with power settings of 50 KV and 70 mA. A Zr filter is used to eliminate the MoK radiation. A suitable crystal was chosen under a microscope, mounted in a nylon loop and aligned on the goniometer prior to the x-ray experiment. A total of 174 images corresponding to a 2.5 phi rotation were collected at room temperature. The reflections on the diffraction images were indexed and integrated using the Automar data processing suite (MARRESEARCH). During the integration the friedel pairs were kept unmerged in order to preserve the anomalous signal needed for absolute structure determination. Xprep (Bruker) was used to determine the spacegroup and to generate the reflection and instruction files for structure determination and subsequent refinement. Structure solution was performed by SHELXS and the refinement was done by SHELXL (A short history of SHELX. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122). The free rotation around the C6-C1 (CIA or C1B) results in rotational isomerism in a 58/42% ratio as seen in FIG. 1 below. As depicted in the X-ray Figure below the chirality of C22 carbon atom is established as R. (H. D. Flack (1983). On Enantiomorph-Polarity Estimation. Acta Cryst A39: 876-881; J. Appl. Cryst. (2008), 41, 96-103.)

(236) It can be readily appreciated that related compounds of the invention may be synthesized from the ad hoc reagents using the general methods and procedures described herein.

III. X-Ray Crystallographic Characterization

III.1. Compound N.SUP.o .1

(237) Compound n.sup.o 1 was characterized by single crystal X-ray spectroscopy thus establishing the configuration of the more active enantiomer as the (R)-configuration (see FIG. 1).

(238) Methods.

(239) All data were recorded on a MAR345 image plate (MARRESEARCH) using MoK radiation (=0.71073). X-rays were generated on a RIGAKU rotating anode generator with power settings of 50 KV and 70 mA. A Zr filter is used to eliminate the MoK radiation. A suitable crystal was chosen under a microscope, mounted in a nylon loop and aligned on the goniometer prior to the x-ray experiment. A total of 174 images corresponding to a 2.0 phi rotation were collected at room temperature. The reflections on the diffraction images were indexed and integrated using the Automar data processing suite (MARRESEARCH). During the integration the friedel pairs were kept unmerged in order to preserve the anomalous signal needed for absolute structure determination. Xprep (Bruker) was used to determine the spacegroup and to generate the reflection and instruction files for structure determination and subsequent refinement. Structure solution was performed by SHELXS and the refinement was done by SHELXL (A short history of SHELX. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122). The free rotation around the C6-C1 (CIA or C1B) results in rotational isomerism in a 58/42% ratio as seen in FIG. 1 below. As depicted in the X-ray FIG. 1 the chirality of C22 carbon atom is established as R. (H. D. Flack (1983). On Enantiomorph-Polarity Estimation. Acta Cryst A39: 876-881; J. Appl. Cryst. (2008), 41, 96-103.)

III.2. Compound N.SUP.o .19

(240) Compound n.sup.o 19 in the present invention was characterized by single crystal X-ray spectroscopy that established the configuration of the more active enantiomer as (R) (see FIG. 2).

(241) Methods.

(242) All data were recorded on a MAR345 image plate (MARRESEARCH) using MoK radiation (=0.71073). X-rays were generated on a RIGAKU rotating anode generator with power settings of 50 KV and 70 mA. A Zr filter is used to eliminate the MoK radiation. A suitable crystal was chosen under a microscope, mounted in a nylon loop and aligned on the goniometer prior to the x-ray experiment. A total of 103 images corresponding to a 1.5 phi rotation were collected at room temperature. The reflections on the diffraction images were indexed and integrated using the Automar data processing suite (MARRESEARCH). During the integration the friedel pairs were kept unmerged in order to preserve the anomalous signal needed for absolute structure determination. Xprep (Bruker) was used to determine the spacegroup and to generate the reflection and instruction files for structure determination and subsequent refinement. Structure solution was performed by SHELXS and the refinement was done by SHELXL (A short history of SHELX. Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122). The free rotation around the C6-C1 (CIA or C1B) results in rotational isomerism in a 56/44% ratio as seen in FIG. 2 below. As depicted in the X-ray FIG. 2 the chirality of C22 carbon atom is established as R. (H. D. Flack (1983). On Enantiomorph-Polarity Estimation. Acta Cryst A39: 876-881; J. Appl. Cryst. (2008), 41, 96-103.)

IV. Summary of Methods and Reagents Used for the Synthesis of the Compounds of the Invention

(243) Compounds of the invention of general Formula I were synthesized from the ad hoc reagents and intermediates using the general methods and procedures described above. Table 4 hereunder recapitulates the intermediates and general methods used for each compound as well as LCMS analytical data.

(244) TABLE-US-00004 TABLE 4 Chiral HPLC Chiral HPLC LCMS Retention Retention Chiral Triazolo LCMS Retention time(S- time (R- HPLC ee Cpd piperazine Acyl chloride General Purity time LCMS enantiomer) enantiomer) Method (R) no intermediate intermediate method (%) (min) [M + H].sup.+ (min) (min) name (%) 1 II-1 4-(thiophen-2- General 97 3.97 422 11.1 15.3 A 95.3 yl)benzoyl Method M chloride 2 II-1 [1,1- General 97 4.07 416 10.9 14.1 A 93.0 biphenyl]-4- Method M carbonyl chloride 3 3a 4-(thiophen-2- General 95 3.97 408 yl)benzoyl Method G chloride 4 3b 4-(thiophen-2- General 97 4.44 462 yl)benzoyl Method G chloride 5 3j 4-(thiophen-2- General 100 4.31 436 10.0 14.0 A 99.5 yl)benzoyl Method G chloride 6 3j [1,1- General 99 4.34 430 6.5 10.4 B 99.5 biphenyl]-4- Method G carbonyl chloride 7 3k 4-(thiophen-2- General 100 4.28 434 10.0 15.0 A 99.6 yl)benzoyl Method G chloride 8 3k [1,1- General 100 4.36 428 9.9 14.3 A 99.0 biphenyl]-4- Method G carbonyl chloride 9 3c 4-(thiophen-2- General 95 4.13 420 yl)benzoyl Method G chloride 10 3c [1,1- General 95 4.22 414 biphenyl]-4- Method G carbonyl chloride 11 3l 4-(thiophen-2- General 100 3.74 406 10.6 15.2 A 96.0 yl)benzoyl Method G chloride 12 3l [1,1- General 99 3.87 400 10.1 13.6 A 99.4 biphenyl]-4- Method G carbonyl chloride 13 3m 4-(thiophen-2- General 98 4.32 434 9.2 13.5 A 99.9 yl)benzoyl Method G chloride 14 3d 4-(thiophen-2- General 94 4.17 420 yl)benzoyl Method G chloride 15 3n 4-(thiophen-2- General 96 4.14 432 7.0 9.7 B 99.9 yl)benzoyl Method G chloride 16 3o 4-(thiophen-2- General 100 4.13 436 8.2 10.9 A 64 yl)benzoyl Method G chloride 17 3p 4-(thiophen-2- General 98 4.03 451 18.1 14.2 C 96.0 yl)benzoyl Method G chloride furnished aminothiazole 4that was then dimethylated using conven- tional method 18 3e 4-(thiophen-2- General 92 4.36 436 yl)benzoyl Method G chloride 19 3v 4-(thiophen-2- General 100 4.30 422 5.9 6.6 A 94.0 yl)benzoyl Method G chloride 20 3v [1,1- General 99 4.41 416 6.3 8.7 B 99.5 biphenyl]-4- Method G carbonyl chloride 21 3f [1,1- General 98 4.52 402 biphenyl]-4- Method G carbonyl22chloride 22 3q 4-(thiophen-2- General 99 4.52 436 6.6 8.5 B 99.0 yl)benzoyl Method G chloride 23 3r 4-(thiophen-2- General 100 4.01 407 6.5 8.7 B 99.8 yl)benzoyl Method G (3/2/0.5 chloride ratio used) 24 3s 4-(thiophen-2- General 100 4.24 423 6.34 7.73 B- 99.0 yl)benzoyl Method G chloride 25 3u 4-(thiophen-2- General 97 4.84 451 5.3 7.7 B- 93.0 yl)benzoyl Method G chloride 26 3t 4-(thiophen-2- General 100 4.02 406 7.3 10.1 B- 99.6 yl)benzoyl Method G chloride 27 3t [1,1- General 100 4.09 400 6.5 8.5 B 99.9 biphenyl]-4- Method G carbonyl chloride 28 3w 4-(thiophen-2- General 100 3.85 419 7.3 9.2 B 96.0 yl)benzoyl Method G chloride 29 3g [1,1- General 100 4.21 410 3.5 4.7 B 99.9 biphenyl]-4- Method G carbonyl chloride 30 3g 4-(thiophen-2- General 100 4.11 416 4.6 5.4 A 99.0 yl)benzoyl Method G chloride 31 3h 4-(thiophen-2- General 100 4.48 418 8.0 13.4 C 98.0 yl)benzoyl Method G chloride 32 3h [1,1- General 100 3.57 412 7.3 9.0 C 97.0 biphenyl]-4- Method G carbonyl chloride 33 3i 4-(thiophen-2- Method G 99 4.19 427 16.2 17.3 C 98.5 yl)benzoyl chloride

(245) In Table 4, the term Cpd means compound.

(246) In Table 4 the configuration of each peak separated by chiral LC was established with compound n.sup.o 1, compound n.sup.o 19 directly, and applied to the other cases by analogy. The indirect configurational assignment aforesaid was always confirmed through biological activity determination that was conclusive given the acute stereochemical SAR.

BIOLOGY EXAMPLES

(247) Functional Assay

(248) Aequorin Assay with Human NK-3 Receptor

(249) Changes in intracellular calcium levels are a recognized indicator of G protein-coupled receptor activity. The efficacy of compounds of the invention to inhibit NKA-mediated

(250) NK-3 receptor activation was assessed by an in vitro Aequorin functional assay. Chinese Hamster Ovary recombinant cells expressing the human NK3 receptor and a construct that encodes the photoprotein apoaequorin were used for this assay. In the presence of the cofactor coelenterazine, apoaequorin emits a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.

(251) Antagonist Testing

(252) The antagonist activity of compounds of the invention is measured following pre-incubation (3 minutes) of the compound with the cells, followed by addition of the reference agonist (NKA) at a final concentration equivalent to the EC.sub.80 (3 nM) and recording of emitted light (FDSS 6000 Hamamatsu) over the subsequent 90-second period. The intensity of the emitted light is integrated using the reader software. Compound antagonist activity is measured based on the inhibition of the luminescence response to the addition of Neurokinin A.

(253) Inhibition curves are obtained for compounds of the invention and the concentrations of compounds which inhibit 50% of reference agonist response (IC.sub.50) were determined (see results in table 5 below). The IC.sub.50 values shown in table 5 indicate that compounds of the invention are potent NK-3 antagonist compounds.

(254) TABLE-US-00005 TABLE 5 Compound no IC.sub.50 (nM) 1 16 2 28 3 83 4 50 5 3 6 10 7 3 8 7 9 18 10 20 11 34 12 58 13 2 14 47 15 9 16 30 17 7 18 10 19 8 20 11 21 33 22 21 23 33 24 2 25 3 26 12 27 51 28 37 29 18 30 11 31 18

(255) Competitive Binding Assays

(256) The affinity of compounds of the invention for the human NK-3 receptor was determined by measuring the ability of compounds of the invention to competitively and reversibly displace a well-characterized NK3 radioligand.

(257) .sup.3H-SB222200 Binding Competition Assay with Human NK-3 Receptor

(258) The ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist .sup.3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK3 receptor. The membranes were incubated with 5 nM .sup.3H-SB222200 (ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl.sub.2 1 mM/MgCl.sub.2 5 Mm/BSA 0.5%/Saponin 10 g/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of .sup.3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentration that displaced 50% of bound radioligand (IC.sub.50) were determined by linear regression analysis and then the apparent inhibition constant (K.sub.i) values were calculated by the following equation: K.sub.i=IC.sub.50/(1+[L]/K.sub.d) where [L] is the concentration of free radioligand and K.sub.d is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973) (see results in table 6 below).

(259) Table 6 shows biological results obtained using the .sup.3H-SB222200 binding competition assay with compounds of the invention. These results indicate that compounds of the invention display potent affinity for the human NK-3 receptor.

(260) TABLE-US-00006 TABLE 6 Compound no Ki (nM) 1 16 2 26 3 83 4 56 5 5 6 11 7 4 8 7 9 19 10 36 11 44 12 70 13 3 14 42 15 11 16 32 17 7 18 20 19 6 20 12 21 38 22 21 23 29 25 3 27 51 28 68 29 23 30 10 31 22

(261) Selectivity Assay

(262) Selectivity of the compounds of the invention was determined over the other human NK receptors, namely NK-1 and NK2 receptors.

(263) Human NK1

(264) The affinity of compounds of the invention for the NK1 receptor was evaluated in CHO recombinant cells which express the human NK1 receptor. Membrane suspensions were prepared from these cells. The following radioligand: [.sup.3H] substance P (PerkinElmer Cat #NET111520) was used in this assay. Binding assays were performed in a 50 mM Tris/5 mM MnC12/150 mM NaCl/0.1% BSA at pH 7.4. Binding assays consisted of 25 l of membrane suspension (approximately 5 g of protein/well in a 96 well plate), 50 l of compound or reference ligand (Substance P) at increasing concentrations (diluted in assay buffer) and 2 nM [.sup.3H] substance P. The plate was incubated 60 min at 25 C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in 0.5% PEI for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC.sub.50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC.sub.50/(1+[L]/K.sub.D) where [L] is the concentration of free radioligand and K.sub.D is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).

(265) Human NK2

(266) The affinity of compounds of the invention for the NK2 receptor was evaluated in CHO recombinant cells which express the human NK2 receptor. Membrane suspensions were prepared from these cells. The following radioligand [.sup.125I]-Neurokinin A (PerkinElmer Cat #NEX252) was used in this assay. Binding assays were performed in a 25 mM HEPES/1 mM CaCl2/5 mM MgCl2/0.5% BSA/10 g/ml saponin, at pH 7.4. Binding assays consisted of 25 l of membrane suspension (approximately 3.75 g of protein/well in a 96 well plate), 50 l of compound or reference ligand (Neurokinin A) at increasing concentrations (diluted in assay buffer) and 0.1 nM [.sup.125I]-Neurokinin A. The plate was incubated 60 min at 25 C. in a water bath and then filtered over GF/C filters (Perkin Elmer, 6005174, presoaked in assay buffer without saponine for 2 h at room temperature) with a Filtration unit (Perkin Elmer). The radioactivity retained on the filters was measured by using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC.sub.50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC.sub.50/(1+[L]/K.sub.D) where [L] is the concentration of free radioligand and K.sub.D is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).

(267) The compounds of the invention, which were tested in the above NK-1 and NK-2 described assays, demonstrated a low affinity at the human NK-1 and human NK-2 receptors: more than 200 fold shift of the Ki compared to the human NK-3 receptor (table 7). Thus, compounds according to the invention have been shown to be selective over NK1 and NK2 receptors.

(268) TABLE-US-00007 TABLE 7 Compound NK1 Ki NK2Ki NK3 Ki no (M) (M) (nM) 1 12.7 14.0 16 3 >>10 >>10 83 (<10% inhibition at (<10% inhibition at 10 M) 10 M) 11 >>10 >>10 44 (<10% inhibition at (<25% inhibition at 10 M) 10 M) 17 for the racemate: for the racemate: 7 6.06 9.95 23 for the racemate: for the racemate: 29 >>10 >>10 (<10% inhibition at (<25% inhibition at 10 M) 10 M) 30 17.2 5.93 10 31 >>10 >>10 22 (<10% inhibition at (<10% inhibition at 10 M) 10 M)

(269) hERG Inhibition Assay

(270) The human Ether-a-go-go Related Gene (hERG) encodes the inward rectifying voltage gated potassium channel in the heart (I.sub.Kr) which is involved in cardiac repolarisation. I.sub.Kr current inhibition has been shown to elongate the cardiac action potential, a phenomenon associated with increased risk of arrhythmia. I.sub.Kr current inhibition accounts for the vast majority of known cases of drug-induced QT-prolongation. A number of drugs have been withdrawn from late stage clinical trials due to these cardiotoxic effects, therefore it is important to identify inhibitors early in drug discovery.

(271) The hERG inhibition study aims at quantifying the in vitro effects of compounds of the invention on the potassium-selective IK.sub.r current generated in normoxic conditions in stably transfected HEK 293 cells with the human ether-a-go-go-related gene (hERG).

(272) Whole-cell currents (acquisition by manual patch-clamp) elicited during a voltage pulse were recorded in baseline conditions and following application of tested compounds (5 minutes of exposure). The concentrations of tested compounds (0.3 M; 3 M; 10 M; 30 M) reflect a range believed to exceed the concentrations at expected efficacy doses in preclinical models.

(273) The pulses protocol applied is described as follow: the holding potential (every 3 seconds) was stepped from 80 mV to a maximum value of +40 mV, starting with 40 mV, in eight increments of +10 mV, for a period of 1 second. The membrane potential was then returned to 55 mV, after each of these incremented steps, for 1 second and finally repolarized to 80 mV for 1 second.

(274) The current density recorded were normalized against the baseline conditions and corrected for solvent effect and time-dependent current run-down using experimental design in test compound free conditions.

(275) Inhibition curves were obtained for compounds and the concentrations which decreased 50% of the current density determined in the baseline conditions (IC.sub.50) were determined. All compounds for which the IC.sub.50 value is above 10 M are not considered to be potent inhibitors of the hERG channel whereas compounds with IC.sub.50 values below 1 M are considered potent hERG channel inhibitors.

(276) When tested in the hERG inhibition assay, compounds of the invention were determined to have IC.sub.50 values as shown in Table 8.

(277) TABLE-US-00008 TABLE 8 Compound no IC.sub.50 (M) 1 >30 2 >30 3 26 4 >30 11 >30 19 17 22 12 29 25 31 20

(278) In Vivo Assay to Assess Compound Activity in Rat

(279) The effect of compounds of the invention to inhibit luteinizing hormone (LH) secretion and decrease circulating androgen levels are determined by the following biological studies.

(280) Castrated Male Rat Model to Assess the Effect of Compound of Invention on Circulating Levels of Luteinizing Hormone (LH).

(281) In humans and rodents, castration is well-precedented to permit heightened, persistent GnRH signaling and consequent elevation of circulating LH. Thus, a castrated rat model is used to provide a broad index for measurement of LH inhibition as a marker of test compound inhibition of the GnRH signaling pathway.

(282) Castrated adult male Sprague-Dawley (SD) rats (150-175 g,) were purchased from Janvier (St Berthevin, France). All animals were housed 3 per cage in a temperature-controlled room (222 C.) and 505% relative humidity with a 12 hour light/12 hour dark photoperiod (lights off at 6 h 00 pm). The animals were allowed 2 weeks of postoperative recovery prior to study. Animals were handled on a daily basis. Standard diet and tap water were provided ad libitum. Animal cage litters were changed once a week. On the study day, animals were acclimated to the procedure room for a period of one hour prior to the initiation of the experiment.

(283) Compounds of the invention were formulated as a pyrogen water with 90 g/L (2-Hydroxypropyl)--CycloDextrin.

(284) After basal sampling (T0) a single dose of compounds of the invention or vehicle was administrated intravenously to rats. Blood was then collected at 60 min post dosing. Blood samples were obtained via tail vein bleed, drawn into EDTA-containing tubes and centrifuged immediately. Plasma samples were collected and stored in a 80 C. freezer until assayed. Serum LH levels were determined using radioimmunoas say kit from RIAZENRat LH, Zentech (Lige, Belgium). Baseline was defined as the initial basal blood sample.

(285) When tested in the castrated male rat model described above, the compound n.sup.o 1 significantly suppressed circulating LH levels (FIG. 3).

(286) When tested in the castrated male rat model described above, the compound n.sup.o 19 significantly suppressed circulating LH levels (FIG. 4).

(287) Gonad-Intact Adult Male to Assess the Effect of Compounds of the Invention on Circulating Levels of Testosterone.

(288) Gonad-intact adult male Sprague-Dawley (SD) rats (225-385 g N=3/group were housed in a temperature-controlled room (222 C.) and 505% relative humidity with a 12 hour light/12 hour dark photoperiod (lights off at 6 h 00 pm). Rat chow and tap water were made available to rats, ad libitum. After basal blood sampling, free-moving rats were intravenously injected at time=0 min with either a single dose of compound or vehicle. Blood was then collected at times 1, 5, 15, 90, 150, 210 min into tubes containing EDTA as anticoagulant and centrifuged immediately. Plasma samples were collected and stored in a 80 C. freezer until assayed. Plasma testosterone levels were determined using a radioimmunoassay kit (Immunotech).

(289) Compound n.sup.o 1 was formulated in 9% 2-hydroxypropyl--cyclodextrin/H2O (w/w). A single dose of 50 mg/kg of compound n.sup.o 1 was intravenously injected.

(290) When tested in gonad-intact male rats, compound n.sup.o 1 significantly suppressed plasma testosterone levels over the 210 minute test period (FIG. 5).